Blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) is a critical tool in the medical and scientific communities. Despite the indispensable role of the BOLD fMRI technique in mapping human brain function, its physiological sources are not well known because the BOLD effect has a complex dependence on many factors including cerebral blood flow (CBF), cerebral blood volume (CBV), and cerebral metabolic rate of O2 consumption (CMRO2). Thus, it is imperative to first perform an in-depth examination of the physiological origins of fMRI signals in order to utilize the fullest capabilities of the technique. During the last grant period, we investigated the spatial specificity of hemodynamic responses, characterized the physiological source of spin-echo BOLD and diffusion fMRI signals, and established a novel approach to maintain evoked neural activity and metabolic responses with negligible functional CBF and CBV changes. In this competitive renewal application, we aim to further elucidate the sources of both BOLD and CBV fMRI responses using a well-established animal model, since its neural properties are similar to humans (e.g., existence of visual columns and retinotopic organization). Our detailed goals are 1) to determine contributions from various physiological sources of the commonly-observed post-stimulus BOLD undershoot in order to test the hypothesis that the post-stimulus BOLD undershoot has major contributions from sustained oxygen consumption changes, 2) to determine specificity to sites of neural activity for the late CBV response vs. the early CBV response in order to test the hypothesis that the late response has improved spatial specificity, 3) to determine relationships between changes in CBF, arterial CBV, and venous CBV during increased neural activity and hypercapnia in order to test the hypothesis that the current assumptions for quantification of CMRO2 changes are not valid, and 4) to determine the impact of anesthesia on neural activity-induced hemodynamic responses in order to test the hypothesis that conclusions drawn from studies of anesthetized animals can be used to explain awake-state functional data. The long-term goal of these investigations is to determine the detailed physiological mechanisms responsible for BOLD fMRI responses, and the time-dependent spatial specificity of various fMRI signals. These investigations involving fMRI and physiology measurements will provide an understanding of the spatiotemporal relationships between BOLD, CBF, arterial blood volume, and venous blood volume during neural activation. Consequently, BOLD signals can be interpreted and quantified as more meaningful physiological parameters in normal subjects and patients. PUBLIC HEALTH RELEVANCE: Blood oxygenation level dependent (BOLD) fMRI is a critical tool in the medical and scientific communities, but its physiological sources are not well known. Proposed investigations aim to interpret and quantify the commonly-used BOLD functional MRI signals as meaningful physiological parameters in normal subjects and patients.